Crosslinked elastin and process for producing the same

ABSTRACT

A crosslinked elastin, a water-soluble crosslinking agent to be used for crosslinking, molded elastin articles, medical instruments and regeneration tissues using the crosslinked elastin, and a surgical therapy method and regeneration treatment wherein the medical instruments are employed. There is provided a biocompatible functional material having elasticity suitable for transplantation into the body without causing detachment of cell adhesion proteins.

This application claims priority from Application Serial No.2001-163,505, Filed on May 30, 2001 in Japan. This application alsoclaims priority from PCT International Patent Application No.PCT/JP02/05275, Filed on May 30, 2002 and published on Dec. 5, 2002 asWO 02/096978 A1. The contents of the preceeding applications areincorporated herein by reference as fully set forth herein for allpurposes.

FIELD OF THE INVENTION

The present invention relates to a biocompatible functional material,and to its production process, medical instruments, crosslinking agents,surgical therapy methods and regeneration tissue.

DISCUSSION OF THE BACKGROUND ART

One method used to treat patients with severed nerve tissue resultingfrom accidents, disasters and other causes has been to ligate tubes madeof artificial materials to the site of nerve deficiency to induceregeneration of nerve tissue in the tube. The tubes used have interiorsof silicone, polyurethane, polyester, polyethylene terephthalate,alginic acid, polylactic acid or the like, coated with a cell adhesionprotein such as collagen or laminin.

As a therapy method for patients with severed blood vessels, a fabriccomposed of knitted synthetic polymer fibers made of silicone,polyurethane, polyester or the like is formed into a tube, and theinside is coated with a cell adhesion protein such as collagen orlaminin to prepare an artificial blood vessel which is transplanted intothe site of the severed blood vessel to induce endothelial cells intothe artificial blood vessel.

SUMMARY OF THE INVENTION

In the therapy method described above, the silicone tubes orpolyurethane tubes or artificial blood vessels are coated with celladhesion proteins such as collagen or laminin, but since the innersurfaces of the tubes have no cell adhesion properties, the celladhesion proteins become detached with extended therapy resulting ininadequate regeneration of the nerve or blood vessel tissue.

Tubes or artificial blood vessels transplanted into an animal must beelastic so as to follow the movement of the body or tissue, but becausetubes or artificial blood vessels composed mainly of silicone orpolyester have a Young's modulus (elastic modulus) of 1×10⁷ Pa orgreater as compared to the Young's modulus (elastic modulus) of 1×10⁴ to2×10⁶ Pa for the accommodating tissue, the joint is therefore subjectedto high stress and can lead to such problems as thrombosis. Materialswith the same elasticity as human tissue have therefore been desired.

As a result of diligent research conducted in the light of theseproblems of the prior art, the present inventors have found that bycrosslinking a water-soluble elastin with a crosslinking agent it ispossible to obtain a crosslinked elastin having elasticity suitable fortransplantation into the body while avoiding detachment of the coatedcell adhesion proteins such as collagen or laminin, and the presentinvention has been completed on the basis of this finding.

The invention has the following construction.

(1) A crosslinked elastin comprising a crosslinking starting materialcontaining at least one type of water-soluble elastin crosslinked with awater-soluble crosslinking agent.

(2) A crosslinked elastin according to (1), wherein the crosslinkingstarting material further comprises one or more components selected fromamong proteins such as collagen, gelatin, fibronectin, fibrin, laminin,casein, keratin, sericin and thrombin, polyamino acids such aspolyglutamic acid and polylysine, sugars such as polygalacturonic acid,heparin, chondroitin sulfate, hyaluronic acid, dermatan sulfate,chondroitin, dextran sulfate, sulfated cellulose, alginic acid, dextran,carboxymethylchitin, galactomannan, gum arabic, tragacanth gum, gelangum, sulfated gelan, karaya gum, carrageenan, agar, xanthan gum,curdlan, pullulan, cellulose, starch, carboxymethyl cellulose, methylcellulose, water-soluble soybean polysaccharide, glucomannan, chitin,chitosan, xyloglucan and lentinan, cell growth factors such as bFGF(basic Fibroblast Growth Factor), TGF-α (Transforming Growth Factor α),EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor)and CNTF (Ciliary NeuroTrophic Factor), as well as polymethylmethacrylate, polydimethylsiloxane, polytetrafluoroethylene, silicone,polyurethane, polyethylene terephthalate, polypropylene, polyethylene,polycaprolactone, polypropylene ether, polytetramethylene glycol,polyethylene glycol, polylactic acid, polyvinyl alcohol and polymalicacid.

(3) A crosslinked elastin according to (1), wherein the water-solubleelastin content is in the range of 0.5–99.5 wt %.

(4) A crosslinked elastin according to (1), wherein the Young's modulusis in the range of 1×10² to 1×10⁷ Pa.

(5) A crosslinked elastin according to (1), wherein the internalstructure is a porous sponge structure.

(6) A crosslinked elastin according to (5), wherein the mean diameter ofthe pores is less than 20 μm.

(7) A crosslinked elastin according to (5), wherein the mean diameter ofthe pores is in the range of 20 μm to 2 mm.

(8) A crosslinked elastin according to (1) or (2), wherein the one ormore components selected from among proteins such as collagen, gelatin,fibronectin, fibrin, laminin, casein, keratin, sericin and thrombin,polyamino acids such as polyglutamic acid and polylysine, sugars such aspolygalacturonic acid, heparin, chondroitin sulfate, hyaluronic acid,dermatan sulfate, chondroitin, dextran sulfate, sulfated cellulose,alginic acid, dextran, carboxymethylchitin, galactomannan, gum arabic,tragacanth gum, gelan gum, sulfated gelan, karaya gum, carrageenan,agar, xanthan gum, curdlan, pullulan, cellulose, starch, carboxymethylcellulose, methyl cellulose, water-soluble soybean polysaccharide,glucomannan, chitin, chitosan, xyloglucan and lentinan, cell growthfactors such as bFGF (basic Fibroblast Growth Factor), TGF-α(Transforming Growth Factor α), EGF (Epidermal Growth Factor), VEGF(Vascular Endothelial Growth Factor) and CNTF (Ciliary NeuroTrophicFactor), as well as polymethyl methacrylate, polydimethylsiloxane,polytetrafluoroethylene, silicone, polyurethane, polyethyleneterephthalate, polypropylene, polyethylene, polycaprolactone,polypropylene ether, polytetramethylene glycol, polyethylene glycol,polylactic acid, polyvinyl alcohol and polymalic acid, are chemicallybonded.

(9) A crosslinked elastin according to (8), wherein the chemical bond isa crosslink produced using a crosslinking agent.

(10) A crosslinked elastin according to (1), (2) or (8), which comprisesone or more components selected from among proteins such as collagen,gelatin, fibronectin, fibrin, laminin, casein, keratin, sericin andthrombin, polyamino acids such as polyglutamic acid and polylysine,sugars such as polygalacturonic acid, heparin, chondroitin sulfate,hyaluronic acid, dermatan sulfate, chondroitin, dextran sulfate,sulfated cellulose, alginic acid, dextran, carboxymethylchitin,galactomannan, gum arabic, tragacanth gum, gelan gum, sulfated gelan,karaya gum, carrageenan, agar, xanthan gum, curdlan, pullulan,cellulose, starch, carboxymethyl cellulose, methyl cellulose,water-soluble soybean polysaccharide, glucomannan, chitin, chitosan,xyloglucan and lentinan, cell growth factors such as bFGF (basicFibroblast Growth Factor), TGF-α (Transforming Growth Factor α), EGF(Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor) andCNTF (Ciliary NeuroTrophic Factor), as well as polymethyl methacrylate,polydimethylsiloxane, polytetrafluoroethylene, silicone, polyurethane,polyethylene terephthalate, polypropylene, polyethylene,polycaprolactone, polypropylene ether, polytetramethylene glycol,polyethylene glycol, polylactic acid, polyvinyl alcohol and polymalicacid.

(11) A crosslinked elastin according to (1), wherein the water-solublecrosslinking agent is a water-soluble compound having a hydrophobicportion in the center region of the molecule and having an aminogroup-reacting active ester group at both ends.

(12) A crosslinked elastin according to (1), characterized in that thewater-soluble crosslinking agent is a water-soluble compound representedby the following general formula.

[wherein R₁ and R₃ are each <A> or <B> represented by the followingstructural formulas and R₁ and R₃ may be the same or different:

(wherein R₄ and R₅ are each H, CH₃ or C₂H₅, and R₄ and R₅ may be thesame or different);

and R₂ is <C> or <D> represented by the following structural formulas:<C>

CH₂

_(n)(wherein n is an integer of 1–20);

(wherein m and 1 are each an integer of 0–15, X and Y are each CH₂ or Oand X and Y may be the same or different, Z is C or N, and R₆, R₇, R₈and R₉ are each H, CH₃ or C₂H₅ and may be the same or different).]

(13) A molded elastin article made from a crosslinked elastin accordingto any one of (1) to (12).

(14) A molded elastin article according to (13), wherein the shape isfilamentous, membranous, cylindrical, pelleted or tubular.

(15) A medical instrument employing a crosslinked elastin according to(1).

(16) A surgical therapy method characterized by utilizing a medicalinstrument according to (15).

(17) A regeneration treatment characterized by utilizing a crosslinkedelastin according to (1) or a medical instrument according to (15).

(18) Regeneration tissue obtained using a crosslinked elastin accordingto (1).

(19) A crosslinking agent comprising a water-soluble compound having ahydrophobic portion in the center region of the molecule and having anamino group-reacting active ester group at both ends.

(20) A crosslinking agent according to (19), wherein the compound is acompound represented by the following general formula.

[wherein R₁ and R₃ are each <A> or <B> represented by the followingstructural formulas and R₁ and R₃ may be the same or different:

(wherein R₄ and R₅ are each H, CH₃ or C₂H₅, and R₄ and R₅ may be thesame or different);

and R₂ is <C> or <D> represented by the following structural formulas:<C>

CH₂

_(n)(wherein n is an integer of 1–20);

(wherein m and 1 are each an integer of 0–15, X and Y are each CH₂ or Oand X and Y may be the same or different, Z is C or N, and R₆, R₇, R₈and R₉ are each H, CH₃ or C₂H₅ and may be the same or different).]

(21) A production process of crosslinked elastin, characterized bycrosslinking a water-soluble elastin by crosslinking reaction using awater-soluble crosslinking agent according to (19).

(22) A production process of crosslinked elastin according to (21),characterized in that the reaction temperature for the crosslinkingreaction is in the range of 4–150° C.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is an electron micrograph of the crosslinked elastin of theinvention according to Example 8 (25° C. reaction).

FIG. 2 is an electron micrograph of the crosslinked elastin of theinvention according to Example 9 (50° C. reaction).

FIG. 3 is an image showing the 1% elastin/gelatin molded crosslinkedarticle of Example 10.

FIG. 4 is an image showing the 10% elastin/gelatin molded crosslinkedarticle of Example 11.

FIG. 5 is an image showing the 90% elastin/gelatin molded crosslinkedarticle of Example 12.

FIG. 6 is an image showing the 0% elastin/gelatin molded crosslinkedarticle of Example 13.

FIG. 7 is a set of comparative images of 0–90% elastin/gelatin moldedcrosslinked articles.

FIG. 8 is an image showing the heparin-containing elastin moldedcrosslinked article of Example 14.

FIG. 9 is a set of images showing the heparin content confirming test ofExample 15.

FIG. 10 is an image showing the sheet-like crosslinked elastin ofExample 16.

FIG. 11 is an image showing the sheet-like crosslinked elastin ofExample 17.

FIG. 12 is an image showing the filamentous crosslinked elastin ofExample 17.

FIG. 13 is an image showing the pelleted crosslinked elastin of Example17.

FIG. 14 is an image showing the fibroblast growth factor-containingcrosslinked elastin/heparin of Example 19.

FIG. 15 is an image showing the growth curves of neuroblastomas (IMR-32)on cell adhesion proteins. Δ: gelatin, ⊚: elastin, □: albumin, No:initial cell count on protein-coated culturing plate, Nt: measured cellcount.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The water-soluble elastin used for the invention is not particularlyrestricted and may be obtained by hydrolysis. Specifically, there may beused at least one type of elastin selected from among α-elastin orβ-elastin obtained by hot oxalic acid treatment of animal cervicalligament or the like, K-elastin obtained by alkali ethanol treatment ofelastin, water-soluble elastin obtained by enzyme treatment withelastase, and tropoelastin which is the precursor in the elastinbiosynthesis pathway. There are no particular restrictions ontropoelastin, and it may be any one or more types from among extractsfrom animal cells and tropoelastin gene products obtained by generecombination.

Elastin is an elastic protein normally found in the body, and abundantlyfound in body tissues which require elasticity, such as arteries andvocal cords. Elastin present in the body is water-insoluble due to ahigh content of hydrophobic amino acids and rigid crosslinked structuressuch as desmosine and isodesmosine. Such elastin exhibits an elasticproperty as a result of unique structures known as “oiled coils” createdby the crosslinking.

The crosslinked elastin of the present invention may be obtained byusing a water-soluble crosslinking agent to crosslink one or more typesof water-soluble elastin rendered water-soluble by degradation of thecrosslinked structure of biogenic elastin. Molded elastin articles ofthe invention may be obtained by combining the aforementionedwater-soluble elastin and water-soluble crosslinking agent to prepare awater-soluble aqueous elastin solution, and then casting it onto amolding template or the like, heating and so forth to accomplishcrosslinking.

The crosslinked elastin of the invention may also comprise a thirdcomponent in addition to the water-soluble elastin and crosslinkingagent. Such a third component is not particularly restricted. Asexamples of third components there may be mentioned proteins such ascollagen, gelatin, fibronectin, fibrin, laminin, casein, keratin,sericin and thrombin, polyamino acids such as polyglutamic acid andpolylysine, sugars such as polygalacturonic acid, heparin, chondroitinsulfate, hyaluronic acid, dermatan sulfate, chondroitin, dextransulfate, sulfated cellulose, alginic acid, dextran, carboxymethylchitin,galactomannan, gum arabic, tragacanth gum, gelan gum, sulfated gelan,karaya gum, carrageenan, agar, xanthan gum, curdlan, pullulan,cellulose, starch, carboxymethyl cellulose, methyl cellulose,water-soluble soybean polysaccharide, glucomannan, chitin, chitosan,xyloglucan and lentinan, cell growth factors such as bFGF (basicFibroblast Growth Factor), TGF-α (Transforming Growth Factor α), EGF(Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor) andCNTF (Ciliary NeuroTrophic Factor), as well as compounds such aspolymethyl methacrylate, polydimethylsiloxane, polytetrafluoroethylene,silicone, polyurethane, polyethylene terephthalate, polypropylene,polyethylene, polycaprolactone, polypropylene ether, polytetramethyleneglycol, polyethylene glycol, polylactic acid, polyvinyl alcohol andpolymalic acid. Incorporation of one or more such components will causeno problem. Particularly preferred are extracellular matrix componentssuch as collagen, gelatin, fibronectin, laminin, heparin and chondroitinsulfate or cell growth factors such as bFGF (basic Fibroblast GrowthFactor), for enhanced cell adhesion and growth.

The proportion of water-soluble elastin in the crosslinked elastin ofthe invention is preferably in the range of 0.5–99.5 wt % with respectto the crosslinked elastin. The range is more preferably 1–95%, sincethis range will give satisfactorily moldable molded articles withbiocompatible elasticity.

The water-soluble elastin is a hydrophobic protein of whichapproximately 94% of the total weight consists of hydrophobic aminoacids and approximately 1% consists of amino acids with amino groups onthe side chain (lysine, arginine, histidine). The water-solublecrosslinking agent used for the invention may be any water-solublecrosslinking agent which reacts with the side chain amino groups of thewater-soluble elastin to accomplish crosslinking reaction. As examplesof water-soluble crosslinking agents there may be mentionedglutaraldehyde, ethyleneglycidyl ether and the like, or compounds havinga hydrophobic portion in the center region of the molecule and having anactive ester group at both ends, as represented by the general formulashown below. A compound represented by the following general formula ispreferably used as the crosslinking agent in order to yieldsatisfactorily shapeable molded articles with biocompatible elasticity.

[wherein R₁ and R₃ are each <A> or <B> represented by the followingstructural formulas and R₁ and R₃ may be the same or different:

(wherein R₄ and R₅ are each H, CH₃ or C₂H₅, and R₄ and R₅ may be thesame or different);

R₂ is <C> or <D> represented by the following structural formulas:

<C>

CH₂

_(n)(wherein n is an integer of 1–20);

(wherein m and 1 are each an integer of 0–15, X and Y are each CH₂ or 0and X and Y may be the same or different, Z is C or N, and R₆, R₇, R₈and R₉ are each H, CH₃ or C₂H₅ and may be the same or different).]

Compounds having a hydrophobic portion in the center region of themolecule form rigid, stable structures with hydrophobic amino acid-richelastin by hydrophobic interaction. However, although compounds withnumerous hydrophobic portions are soluble in organic solvents, they aresparingly or totally insoluble in water and thus poorly manageable inaqueous systems. The water-soluble crosslinking agent of the inventionis characterized by having both ends of the dicarboxylic acid compoundrepresented by the above-mentioned general formula converted to anactive ester with 4-hydroxyphenyldimethyl-sulfonium methylsulfate (DSP),and by having a hydrophobic portion which forms a rigid, stablestructure with hydrophobic amino acid-rich elastin, while dissolving inwater and thus being manageable in an aqueous system.

The active ester groups at both ends in the chemical formula of thewater-soluble crosslinking agent of the invention form peptide bondswith amino acids of the water-soluble elastin, thus producingcrosslinks. The crosslinked elastin obtained by crosslinking with thewater-soluble crosslinking agent of the invention is thereforecharacterized by being highly biodegradable in the body. Thebiodegradation rate depends on the degree of crosslinking of thecrosslinked elastin, and may therefore be controlled by varying thecrosslinking conditions to alter the degree of crosslinking.

The structure of the crosslinked elastin of the invention is notparticularly restricted, but is preferably a porous sponge structure soas to allow penetration of body fluids, culture solutions and the like.Although there are no particular restrictions on the sizes of the pores,a mean diameter of less than 20 μm will tend to yield a hard crosslinkedproduct with a high Young's modulus (elastic modulus). On the otherhand, a range of 20 μm to 2 mm will tend to yield a moldable crosslinkedproduct with a low Young's modulus (elastic modulus) and a high degreeof swelling.

The crosslinked elastin of the invention is a highly elastic crosslinkedproduct, and for greater biocompatibility it preferably has a Young'smodulus (elastic modulus) in the range of 1×10² to 1×10⁷ Pa, and morepreferably in the range of 1×10³ to 2×10⁶ Pa.

There are no particular restrictions on the shape of a molded elastinarticle according to the invention, but for medical applications it ispreferably filamentous, membranous, cylindrical, pelleted or tubular.

The crosslinked elastin of the invention may form a specific structureby itself, or it may form a complex with components other than thecrosslinked elastin. Surface coatings with structures other than that ofthe crosslinked elastin may also be used. Components forming suchcomplexes are not particularly restricted, and as examples there may bementioned proteins such as collagen, gelatin, fibronectin, fibrin,laminin, casein, keratin, sericin and thrombin, polyamino acids such aspolyglutamic acid and polylysine, sugars such as polygalacturonic acid,heparin, chondroitin sulfate, hyaluronic acid, dermatan sulfate,chondroitin, dextran sulfate, sulfated cellulose, alginic acid, dextran,carboxymethylchitin, galactomannan, gum arabic, tragacanth gum, gelangum, sulfated gelan, karaya gum, carrageenan, agar, xanthan gum,curdlan, pullulan, cellulose, starch, carboxymethyl cellulose, methylcellulose, water-soluble soybean polysaccharide, glucomannan, chitin,chitosan, xyloglucan and lentinan, cell growth factors such as bFGF(basic Fibroblast Growth Factor), TGF-α (Transforming Growth Factor α),EGF (Epidermal Growth Factor), VEGF (Vascular Endothelial Growth Factor)and CNTF (Ciliary NeuroTrophic Factor), as well as compounds such aspolymethyl methacrylate, polydimethylsiloxane, polytetrafluoroethylene,silicone, polyurethane, polyethylene terephthalate, polypropylene,polyethylene, polycaprolactone, polypropylene ether, polytetramethyleneglycol, polyethylene glycol, polylactic acid, polyvinyl alcohol andpolymalic acid. Any one or more of these may be used to conferbiofunctional properties such as cell adhesion or antithromboticactivity not possessed by the elastin, or to increase the growth rate ofthe tissue of interest.

The conditions for the crosslinking reaction between the water-solubleelastin and water-soluble crosslinking agent are not particularlyrestricted, but the reaction temperature is preferably in the range of4–150° C. at ordinary pressure or under pressurization with an autoclaveor the like. A range of 10–120° C. is particularly preferred from thestandpoint of manipulation of the crosslinking. When the crosslinkedelastin of the invention has a porous sponge structure, the reactiontemperature may be controlled to allow control of the pore diameters.For example, a reaction temperature in the range of 4–50° C. can yieldcrosslinked products with pore mean diameters of 20 μm and greater,while a temperature in the range of 50–150° C. can yield crosslinkedproducts with pore mean diameters of less than 20 μm.

The method of molding the crosslinked elastin of the invention is notparticularly restricted, and a molding die commonly used for molding ofsynthetic resins may be used. For example, the water-soluble elastin andthe water-soluble crosslinking agent of the invention may be combinedand the resulting water-soluble elastin aqueous solution may be castinto a molding machine and heated with an autoclave or the like forcrosslinking, to produce crosslinked elastin having a filamentous,membranous, cylindrical, pelleted or tubular shape reflecting that ofthe die.

Because the crosslinked elastin of the invention has elasticity in thesame range as that of living tissue, it has excellent stretchability andcan be effectively used for cosmetics and medical instruments. It is notparticularly restricted in its uses for cosmetics and may serve, forexample, as a face mask base for skin care products. There are also noparticular restrictions on its uses for medical instruments, andapplication for molding of parts such as catheters, shunts, woundcoverings and the like may provide more flexible functions than haveexisted in the prior art.

When a medical instrument of the invention employing such materials asregeneration treatment materials is implanted into the body, the tissueof interest will grow more favorably in the body. Such use isparticularly effective for neurons and blood vessels, as mentionedabove.

In order to further increase the cell growth rate and improvebiocompatibility, the crosslinked elastin may be combined with thirdcomponents to confer functions not naturally exhibited by elastin. Forexample, there may be included cell growth factors or heparin, which isantithrombotic and interacts with cell growth factors.

Such third components may be premixed with the starting material duringformation of the crosslinked elastin for incorporation into thecrosslinked product, or the crosslinked elastin may be formed first andthe third components impregnated into the structure or physicallyadsorbed onto it after drying. Alternatively, the third components maybe chemically anchored to the crosslinked elastin in order to inhibittheir loss.

The crosslinked elastin of the invention may be used as a sustainedrelease carrier, which is a type of drug delivery system (DDS). Inparticular, the crosslinked elastin of the invention can yield poroussponge structures with a high Young's modulus (elastic modulus), and canexhibit a curative effect on nerves, blood vessels and the like.

A medical instrument according to the invention has the function andeffect described above, and is therefore particularly effective whenused for surgical therapy. For example, collagen sheets havingcoagulants such as fibrin or thrombin anchored on one side thereof arecurrently used as postoperative hemostatic adhesives. Here, sites ofvisceral hemorrhage created by surgery are covered with the sheets, withthe goal of achieving the synergetic effect of hemostasis by bloodclotting components and hemostasis by the physical compression with thecollagen sheet, as well as greater handling ease, but collagen sheetshave poor stretchability and have therefore exhibited low contactbonding properties when utilized in vigorously moving organs such as theheart. By using a crosslinked elastin sheet according to the invention,however, it is possible to produce a sheet with high stretchabilitywhich can adapt to organ movement. In addition, a complex between thecrosslinked elastin of the invention and collagen may be formed toproduce a very highly biocompatible sheet characterized by exhibitingboth the stretchability of elastin and the cell adhesion properties ofcollagen.

While the crosslinked elastin of the invention may be implanted in thebody as described above for utilization as a scaffold for regenerationof blood vessels or nerves, its function can also be utilized outside ofthe body. Specifically, it may be used as a culturing substrate forregeneration medicine, whereby organs of a desired form can be formed bytransplantation and culturing of direction-oriented embryonic stem (ES)cells, somatic stem cells, mesenchymal stem cells and the like either ona sheet surface or inside a tube according to the invention. Thecrosslinked elastin of the invention not only has satisfactorymoldability but is also biodegradable, and therefore is useful forproviding a cartridge-type regeneration treatment method andregeneration tissue whereby organs which have been partly cultured toshape formation may be transplanted together with the culturingsubstrate.

EXAMPLES

The present invention will now be explained in greater detail throughthe following examples. Unless otherwise specified, the “%” values arebased on weight.

Example 1 Preparation of Water-Soluble Elastin

After adding 150 ml of 0.25 M oxalic acid to 20 g of powderedwater-insoluble elastin (product of Elastin Products Company, Inc), themixture was treated at 100° C. for 1 hour. After cooling, it wascentrifuged (3000 rpm, 30 min) to separation, the supernatant wascollected and placed in a cellulose dialysis tube (molecular cutoff:6000–10,000), and dialysis was performed for 48 hours against deionizedwater to remove the oxalic acid. This was followed by lyophilization toobtain water-soluble elastin. The amino acid analysis results for theelastin starting material and the water-soluble elastin are shown inTable 1.

TABLE 1 Elastin Water-soluble elastin (mol %) (mol %) Aspartic acid0.612 0.542 Threonine 0.778 0.983 Serine 0.665 0.964 Glutamic acid 1.6681.923 Glycine 33.22 30.869 Alanine 22.78 25.512 Cysteine 0.376 0.562Valine 13.374 12.652 Isoleucine 2.568 2.249 Leucine 6.235 6.001 Tyrosine0.703 0.976 Phenylalanine 2.967 3.023 Lysine 0.279 0.381 Histidine 0.0370.063 Arginine 0.516 0.68 Hydroxyproline 1.168 1.007 Proline 11.69911.612 Total 100.00 100.00

Example 2 Production of Crosslinked Elastin Using Glutaraldehyde asCrosslinking Agent

A 90 mg portion of the water-soluble elastin obtained in Example 1 wasadded to and dissolved in 161 μl of deionized water to obtain awater-soluble elastin aqueous solution. To this aqueous solution therewas added 48.7 μl of 250 mM aqueous glutaraldehyde (product of TokyoKasei Kogyo Co., Ltd.), immediately producing a crosslinked elastin gel.It was attempted to cast the crosslinked elastin gel into a cylindricalmolding template with a 2 mm diameter and 2 cm length, but the fluiditywas too poor to allow casting. Casting into the template was difficulteven when the amount of 250 mM aqueous glutaraldehyde addition wasreduced to 1/10 the previous amount.

Example 3 Production of Crosslinked Elastin Using EthyleneglycolDiglycidyl Ether as Crosslinking Agent

A 36 mg portion of the water-soluble elastin obtained in Example 1 wasadded to and dissolved in 41.6 μl of deionized water to obtain awater-soluble elastin aqueous solution. To this aqueous solution therewas added 42.4 μl of 287 mM aqueous ethyleneglycol diglycidyl ether, andupon thorough mixing, the solution was cast into a cylindrical templatewith a 2 mm diameter and 2 cm length and heated at 50° C. for one hourto obtain a crosslinked elastin gel. The obtained crosslinked elastingel was thoroughly washed with deionized water, and upon conducting asimple stretching test by pulling with the fingers at both ends, it wasfound to be fragile against stretching deformation.

Example 4 Production of Crosslinked Elastin Using Water-SolubleCarbodiimide as Crosslinking Agent

A 50 mg portion of the water-soluble elastin obtained in Example 1 wasadded to and dissolved in 10.4 μl of deionized water to obtain awater-soluble elastin aqueous solution. To this aqueous solution therewas added 10.4 μl of 274 mM water-soluble carbodiimide (WSCD, product ofPeptide Institute, Inc.), and after further adding 24.4 μl of 645 mMadipic acid the mixture was thoroughly agitated to obtain a 30% aqueouselastin solution. Long-chain dicarboxylic acids do not dissolve indeionized water (they will dissolve under alkali conditions but then arehighly reactive with carbodiimide), and therefore incomplete dissolutionand partial solidification occurred as expected, making it impossible toaccomplish casting in different templates (glass tubes, membrane-formingdies, etc.).

Example 5 Production of Crosslinked Elastin Using PhotoreactiveSuccinimide Ester as Crosslinking Agent

A 13 mg portion of the water-soluble elastin obtained in Example 1 and 4mg of a photoreactive succinimide ester (NHS-ASA:N-hydroxysuccinimidyl-4-azidosalicylic acid, product of PIERCE) wereadded to and dissolved in 1 ml of deionized water and reacted at roomtemperature for one day. After completion of the reaction, the unreactedportion was removed and the product was purified to obtain 7 mg ofphotoreactive elastin. To this there was added 20 μl of deionized waterto prepare a photoreactive aqueous elastin solution, and 365 nmultraviolet (UV) irradiation for 90 minutes produced a gel. The obtainedgel was thoroughly washed with deionized water. As a result, because thecrosslinking agent was water-insoluble it exhibited low reactivity,while the elastin was insolubilized in the organic solvent and thus alsoexhibited poor reactivity. With photoirradiation, the UV rays penetratedwith a low aqueous elastin solution concentration (a few percent), butat concentrations of 10% or greater, reaction only occurred on the lightirradiation surface and therefore the gelling was poor and reaction wasvirtually impossible in a template.

Example 6 Production of Crosslinked Elastin Using Adipic AcidSuccinimide Ester as Crosslinking Agent

After adding and dissolving 60 mg of the water-soluble elastin obtainedin Example 1 in 119 μl of deionized water, 21 μl of 385 mM aqueousadipic acid succinimide ester was added, and the solution was cast intoa cylindrical template with a 2 mm diameter and 2 cm length and heatedat 80° C. for one hour to obtain a gel. The produced gel was weak (itdid not form the shape of the template), possibly due to partialinsolubilization of the crosslinking agent. The dodecanedicarboxylicacid succinimide ester was insoluble in water and was thereforeunreactive.

Example 7 Production of Water-Soluble Crosslinking Agent [A]

The carboxyl groups of a dicarboxylic acid were converted to activeesters with 4-hydroxyphenyldimethylsulfonium methylsulfate (DSP). Theactive esterification with DSP was carried out in the followingexperiment following a method reported in the field of peptide chemistry(K. Kouge, T. Koizumi, H. Okai and T. Kato (1987) Bull. Chem. Soc. Jpn.,60, 2409 (Journal of the Chemical Society of Japan)).

Dodecanedicarboxylic acid (2.5 mmol) and DSP (5 mmol) were dissolved inacetonitrile (35 ml) at 60° C., and after cooling,dicyclohexylcarbodiimide (DCC) (5 mmol) was added and the mixture wasstirred at 25° C. for 5 hours. The dicyclohexylurea (DC-Urea) producedduring the reaction was removed by filtration with a glass filter. Thereaction mixture (filtrate) was added dropwise to ether (70 ml) tosolidification. The solid was dried under reduced pressure to obtain 1.4g of water-soluble crosslinking agent [A] of the invention. The purityof the obtained crosslinking agent was 98% as determined by ¹H-NMR(JNM-EX-500, JEOL).

Example 8 Fabrication of Molded Elastin Article Using Water-SolubleCrosslinking Agent [A] Obtained in Example 7 as Crosslinking Agent

A 200 mg portion of the water-soluble elastin obtained in Example 1 wasadded to 1 ml of deionized water and the mixture was thoroughly stirredto obtain a 20% water-soluble elastin aqueous solution. The temperatureof the aqueous solution was adjusted to 25° C., 72 μmol of thewater-soluble crosslinking agent [A] obtained in Example 7 was added (at3-fold based on the amino groups (25 μmol) of the elastin in the aqueoussolution), and the mixture was stirred for 5 minutes. Next, 24 μmol oftriethylamine was added, and the mixture was further stirred for 5minutes and then cast into a cylindrical template with a 2 mm diameterand 2 cm length and allowed to stand for 2 days to produce a gel, whichwas then thoroughly washed with deionized water to obtain a milky white,highly elastic, cylindrical molded elastin article. The obtained moldedelastin article was treated in an autoclave at 110° C. for 10 minutes toobtain a sterilized molded elastin article exhibiting no change inshape. Table 2 shows the results of measuring the Young's modulus of themolded elastin article with a tensile strength tester. The measurementwas conducted with the molded elastin article immersed in water. FIG. 1shows a 90× magnification of a cross-section of the obtained moldedelastin article taken with a scanning electron microscope. The electronmicrograph shows that the internal structure of the crosslinked elastinwas a porous sponge structure with pores having a mean diameter of 62μm. The elastic moduli of crosslinked elastin products with differentpores are shown in Table 2

TABLE 2 Gel formation Elastic modulus Elastic modulus temperature (25°C.) (50° C.) 20° C. 1.2–3.5 10³ Pa 1.0–4.0 10³ Pa 50° C. 1.5–7.8 10³ Pa0.7–5.0 10³ Pa

Example 9 Fabrication of Molded Elastin Article Using Water-SolubleCrosslinking Agent [A] Obtained in Example 7 as Crosslinking Agent,Under Different Crosslinking Conditions

Crosslinking reaction and molding were carried out in the same manner asExample 8, except that the temperature of the aqueous solution wasadjusted to 50° C. and the standing time was 6 hours, to obtain a milkywhite, highly elastic, cylindrical molded elastin article. Table 2 showsthe results of measuring the Young's modulus of the molded elastinarticle with a tensile strength tester. The measurement was conductedwith the molded elastin article immersed in water. FIG. 2 shows a 90×magnification of a cross-section of the obtained molded elastin articletaken with a scanning electron microscope. The electron micrograph showsthat the internal structure of the crosslinked elastin was a poroussponge structure with pores having a mean diameter of 9 μm.

Example 10 Production of Crosslinked Elastin with Elastin Content of 1%of Total, and Fabrication of Molded Article)

After adding and dissolving 0.8 mg of the water-soluble elastin obtainedin Example 1 and 72 mg of gelatin in 148 μl of deionized water, 39 μl(278 mM) of the water-soluble crosslinking agent prepared in Example 7was added (the amount of crosslinking agent corresponded to a 2-foldamount with respect to the amino groups of the elastin) to prepare a 30%water-soluble elastin aqueous solution. The solution was cast into amolding die and heated with an autoclave at 120° C. for 30 minutes toobtain crosslinked elastin and a molded article thereof (FIG. 3).

Example 11 Production of Crosslinked Elastin with Elastin Content of 10%of Total, and Fabrication of Molded Article

After adding and dissolving 8 mg of the water-soluble elastin obtainedin Example 1 and 72 mg of gelatin in 148 μl of deionized water, 39 μl(278 mM) of the water-soluble crosslinking agent prepared in Example 7was added (the amount of crosslinking agent corresponded to a 2-foldamount with respect to the amino groups of the elastin) to prepare a 30%water-soluble elastin aqueous solution. The solution was cast into amolding die and heated with an autoclave at 120° C. for 30 minutes toobtain crosslinked elastin and a molded article thereof (FIG. 4).

Example 12 Production of Crosslinked Elastin with Elastin Content of 90%of Total, and Fabrication of Molded Article

After adding and dissolving 72 mg of the water-soluble elastin obtainedin Example 1 and 8 mg of gelatin in 148 μl of deionized water, 39 μl(278 mM) of the water-soluble crosslinking agent prepared in Example 7was added (the amount of crosslinking agent corresponded to a 2-foldamount with respect to the amino groups of the elastin) to prepare a 30%water-soluble elastin aqueous solution. The solution was cast into amolding die and heated with an autoclave at 120° C. for 30 minutes toobtain crosslinked elastin and a molded article thereof (FIG. 5).

Example 13 Production of Crosslinked Gelatin with Elastin Content of 0%of Total, and Fabrication of Molded Article

After adding 148 μl of deionized water to 80 mg of gelatin to dissolveit, 39 μl (278 mM) of the water-soluble crosslinking agent prepared inExample 7 was added (the amount of crosslinking agent corresponded to a2-fold amount with respect to the amino groups of the elastin) toprepare a 30% water-soluble elastin aqueous solution. The solution wascast into a molding die and heated with an autoclave at 120° C. for 30minutes to obtain crosslinked elastin and a molded article thereof (FIG.6).

To summarize the results of Examples 10 to 13, the degree of swellingwas high with an elastin content of 0% (swelling to about 2.3 times thediameter of the template after 6 hours in water), but firm-shaped gelswere only produced beginning with a content of 1% (FIG. 7: elastincontents of 0, 1, 10 and 90%). The degrees of swelling of the other gelswere about 1.5 times the template with the 1% gel, about 1.4 times thetemplate with the 10% gel and about 1.1 times the template with the 90%gel.

The shape stability was higher with a higher elastin content. Uponstretching, the elastic modulus was undetermined but the lower contentgels were more easily torn and exhibited low strength.

The gel is colored in yellow in proportion to the increased content ofthe water-soluble crosslinking elastin solution, which is yellow.

Example 14 Production of Crosslinked Elastin with Sugar Also Containedin the Crosslinking Starting Material, and Fabrication of Molded ElastinArticle

After adding and dissolving 75 mg of the water-soluble elastin obtainedin Example 1 and 5 mg of heparin in 148 μl of deionized water, 39 μl(278 mM) of the water-soluble crosslinking agent prepared in Example 7was added (the amount of crosslinking agent corresponded to a 2-foldamount with respect to the amino groups of the elastin) to prepare a 30%water-soluble elastin aqueous solution. The solution was cast into amolding die and heated with an autoclave at 120° C. for 30 minutes toobtain crosslinked elastin and a molded article thereof (FIG. 8).

Example 15 Confirmation of Heparin Content

After thoroughly washing the prepared gel with deionized water, it wasstained with a 1% Toluidine Blue-O aqueous solution. Toluidine Blue-Ostains from blue to violet upon binding with heparin. As shown in FIG.9, incorporation of heparin in the gel was confirmed.

Example 16 Fabrication of Elastin Membrane

Two Lypersilane-treated (silicone-coated) glass slides were used toprepare a molding die having a silicone rubber sheet as a spacer, and amixed solution comprising the 30% water-soluble elastin aqueous solutionobtained in Example 1 and the water-soluble crosslinking agent preparedin Example 7 (in a 3-fold molar ratio with respect to the elastin aminogroups) was cast without allowing entrance of air or water from theoutside. These conditions were maintained while heat treatment wascarried out in water at 80° C. for 30 minutes, to obtain an elastinmembrane (FIG. 10).

Example 17 Production of Different Molded Elastin Articles

Different molding dies were prepared and a mixed solution comprising the30% water-soluble elastin aqueous solution and the water-solublecrosslinking agent prepared in Example 7 (in a 3-fold molar ratio withrespect to the elastin amino groups) was cast without allowing entranceof air or water from the outside. These conditions were maintained whileheat treatment was carried out in water at 80° C. for 30 minutes, toobtain tubular (FIG. 11), filamentous (FIG. 12) and pelleted (FIG. 13)molded articles.

Example 18 Cell Culturing Method and Growth Rates

An elastin membrane (0.5 mm thickness, 1 cm×1 cm) was placed on aplastic tissue culturing plate (6 wells), and 2 ml of culture solutionwas added thereto and stationed at 37° C. for 30 minutes. The culturesolution was prepared by adding 215 ml of deionized water to 2.57 g ofMEM Hanks' powder to dissolution and then adding 1.17 ml of sodiumbicarbonate solution (7.5%), 2.5 ml of glutamine solution (200 mM) and2.5 ml of non-essential amino acid solution, and finally adding 5 ml ofgentamicin and 25 ml of fetal bovine serum.

Next, 100 μl of neuroblastoma cells (IMR-32, ATCC No. CCL-127) wereseeded therein to a concentration of 1.0×10⁴ cells/ml and incubated for24 hours at 37° C., and the cell count was periodically measured eitherusing a cell counting plate or by direct observation.

As a control, cell growth was evaluated using an albumin coat. Themedium was exchanged daily, and the experiment was carried out threetimes.

(Results) The cell growth rate after the 3rd day from seeding of thecells in the elastin (sheet) was approximately 4-fold. With the albumincoat, the cell growth rate after the 3rd day from seeding of the cellswas only about 1.5-fold. The growth curve is shown in FIG. 15.

Example 19 Elastin Gel Containing Fibroblast Growth Factor (FGF) andHeparin

After adding 148 1 μl of deionized water to 75 mg of water-solubleelastin and 5 mg of heparin to dissolution, 39 μl (278 mM) of thewater-soluble crosslinking agent prepared in Example 7 was added toprepare a 30% elastin aqueous solution. The solution was cast into amolding die and heated at 120° C. (autoclave) for 30 minutes forreaction. The produced gel was washed with a 0.1 M phosphate buffersolution (pH 7.5) and then immersed for 24 hours in a 0.1 M phosphatebuffer solution (pH 7.5) containing 2 μg/ml basic fibroblast growthfactor (bFGF), for adsorption onto the heparin in the gel. The obtainedmolded article is shown in FIG. 14.

INDUSTRIAL APPLICABILITY

The crosslinked elastin of the present invention is a material withelasticity suitable for transplantation into the body without detachmentof coated cell adhesion proteins, and therefore provides an effect whichsolves the problems associated with prior art materials whereby celladhesion proteins become detached with prolonged treatment or wherebyregeneration of tissues such as nerves or blood vessels is inadequate.

Moreover, the water-soluble crosslinking agent of the inventioncrosslinks water-soluble elastin to yield crosslinked elastin withelasticity which permits molding into any shape of a die, and thereforemolded articles may be made into filamentous, membranous, cylindrical,pelleted or tubular shapes or worked into regeneration treatmentmaterials or medical instrument materials, as an effect allowing itsapplication for a wide range of uses.

Furthermore, since the crosslinked elastin of the invention forms aporous sponge structure, it allows permeation of drugs and the like andfacilitates formation of composites with other materials, as an effectto allow provision of new medical materials.

1. A crosslinked elastin comprising a crosslinking starting materialcontaining at least one type of water-soluble elastin crosslinked with awater-soluble crosslinking agent selected from the group consisting ofcompounds represented by the following general formula:

wherein R₁ and R₃ are each A or B represented by the followingstructural formulas and R₁ and R₃ may be the same or different:

wherein R₄ and R₅ are each H, CH₃ or C₂H₅, and R₄ and R₅ may be the sameor different;

and R₂ is C or D represented by the following structural formulas: C

CH₂

_(n) wherein n is an integer from 1–20; and

wherein m and l are each an integer from 0–15, X and Y are each CH₂ or Oand X and Y may be the same or different, Z is C or N, and R₆, R₇, R₈and R₉ are each H, CH₃ or C₂H₅ and may be the same or different.
 2. Acomposition comprising a crosslinked elastin according to claim 1,wherein the crosslinking starting material further comprises at leastone component selected from the group consisting of: collagen, gelatin,fibronectin, fibrin, laminin, casein, keratin, sericin, thrombin,polyglutamic acid, polylysine, polygalacturonic acid, heparin,chondroitin sulfate, hyaluronic acid, dermatan sulfate, chondroitin,dextran sulfate, sulfated cellulose, alginic acid, dextran,carboxymethylchitin, galactomannan, gum arabic, tragacanth gum, gelangum, sulfated gelan, karaya gum, carrageenan, agar, xanthan gum,curdlan, pullulan, cellulose, starch, carboxymethyl cellulose, methylcellulose, water-soluble soybean polysaccharide, glucomannan, chitin,chitosan, xyloglucan, lentinan, bFGF (basic Fibroblast Growth Factor),TGF-α (Transforming Growth Factor α), EGF (Epidermal Growth Factor),VEGF (Vascular Endothelial Growth Factor), CNTF (Ciliary NeuroTrophicFactor), as polymethyl methacrylate, polydimethylsiloxane,polytetrafluoroethylene, silicone, polyurethane, polyethyleneterephthalate, polypropylene, polyethylene, polycaprolactone,polypropylene ether, polytetramethylene glycol, polyethylene glycol,polylactic acid, polyvinyl alcohol and polymalic acid.
 3. A crosslinkedelastin according to claim 1, wherein the water-soluble elastin contentis in the range of 0.5–99.5 wt %.
 4. A crosslinked elastin according toclaim 1, wherein the Young's modulus is in the range of 1×10² to 1×10⁷Pa.
 5. A crosslinked elastin according to claim 1, wherein the internalstructure is a porous sponge structure.
 6. A crosslinked elastinaccording to claim 5, wherein the mean diameter of the pores is lessthan 20 μm.
 7. A crosslinked elastin according to claim 5, wherein themean diameter of the pores is in the range of 20 μm to 2 mm.
 8. Acomposition according to claim 2, wherein the at least one componentselected from the group consisting of collagen, gelatin, fibronectin,fibrin, laminin, casein, keratin, sericin, thrombin, polyglutamic acid,polylysine, polygalacturonic acid, heparin, chondroitin sulfate,hyaluronic acid, dermatan sulfate, chondroitin, dextran sulfate,sulfated cellulose, alginic acid, dextran, carboxymethylchitin,galactomannan, gum arabic, tragacanth gum, gelan gum, sulfated gelan,karaya gum, carrageenan, agar, xanthan gum, curdlan, pullulan,cellulose, starch, carboxymethyl cellulose, methyl cellulose,water-soluble soybean polysaccharide, glucomannan, chitin, chitosan,xyloglucan, lentinan, bFGF (basic Fibroblast Growth Factor), TGF-α(Transforming Growth Factor α), EGF (Epidermal Growth Factor), VEGF(Vascular Endothelial Growth Factor), CNTF (Ciliary NeuroTrophicFactor), polymethyl methacrylate, polydimethylsiloxane,polytetrafluoroethylene, silicone, polyurethane, polyethyleneterephthalate, polypropylene, polyethylene, polycaprolactone,polypropylene ether, polytetramethylene glycol, polyethylene glycol,polylactic acid, polyvinyl alcohol and polymalic acid, are chemicallybonded.
 9. A composition according to claim 8, wherein the chemical bondis a crosslink produced using a crosslinking agent.
 10. A compositioncomprising a crosslinked elastin according to claim 1, furthercomprising at least one component selected from the group consisting of:collagen, gelatin, fibronectin, fibrin, laminin, casein, keratin,sericin, thrombin, polyglutamic acid, polylysine, polygalacturonic acidheparin, chondroitin sulfate, hyaluronic acid, dermatan sulfate,chondroitin, dextran sulfate, sulfated cellulose, alginic acid, dextran,carboxymethylchitin, galactomannan gum arabic, tragacanth gum, gelangum, sulfated gelan, karaya gum, carrageenan, agar, xanthan gum,curdlan, pullulan, cellulose, starch, carboxymethyl cellulose, methylcellulose, water-soluble soybean polysaccharide, glucomannan, chitin,chitosan, xyloglucan lentinan, bFGF (basic Fibroblast Growth Factor),TGF-α (Transforming Growth Factor α), EGF (Epidermal Growth Factor),VEGF (Vascular Endothelial Growth Factor), CNTF (Ciliary NeuroTrophicFactor), polymethyl methacrylate, polydimethylsiloxane,polytetrafluoroethylene, silicone, polyurethane, polyethyleneterephthalate, polypropylene, polyethylene, polycaprolactone,polypropylene ether, polytetramethylene glycol, polyethylene glycol,polylactic acid, polyvinyl alcohol and polymalic acid.
 11. A compositioncomprising a crosslinked elastin according to claim 1, wherein thecrosslinking starting material further comprises at least one componentselected from the group consisting of: proteins, polyaminoacids, sugars,and cell growth factors.
 12. A composition according to claim 11,wherein said at least one component selected from the group consistingof: proteins, polyaminoacids, sugars, and cell growth factors, arechemically bonded.
 13. A composition comprising a crosslinked elastinaccording to claim 1, further comprising at least one component selectedfrom the group consisting of: proteins, polyaminoacids, sugars, and cellgrowth factors, are chemically bonded.